CN105726545A - Cephradine for prevention and/or treatment of TOPK (T-LAK cell-originated protein kinase) activity abnormally increased skin inflammations by inhibition of TOPK - Google Patents
Cephradine for prevention and/or treatment of TOPK (T-LAK cell-originated protein kinase) activity abnormally increased skin inflammations by inhibition of TOPK Download PDFInfo
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Abstract
The invention discloses a novel efficacy and molecular mechanism of cephradine. By combining with the TOPK to inhibit activity of the TOPK and block TOPK signal channels, the cephradine inhibits UV (ultraviolet) induced photodermatoses (such as sunburn and chronic actinic dermatitis). According to immunopathogenesis, expression of TOPK, P38 and JNK (Jun N-terminal kinase) in skin lesions of photodermatoses is increased. According to cell experiments, TOPK activity affect UV induced P38 and JNK phosphorylation. According to molecular docking experiments, the cephradine can be directly combined with TOPK to inhibit TOPK activity, phosphorylationd of P38, JNK and H2AX and secretion of inflammatory factors IL-6 and TNF-alpha.Human and animal experiments verify results of the cell experiments.
Description
The invention belongs to the new pharmacological action of medicine and diseases prevention and treatment field.It is specifically related to cefradine suppression T-LAK origin of cell
The biological activity of protein kinase (TOPK), and cefradine increases prevention and/or treatment by suppression TOPK activity is abnormal
Inflammation and the application of tumor, the Photodermatoses induced including ultraviolet (UV), such as sunburn and farmersskin.
Background technology
Investigation of clinical epidemiology shows, human body is chronically exposed under solar ultraviolet (SUV) can produce scytitis and skin
Tumor, serious threat human health.Ultraviolet radiation in sunlight can be divided three classes according to the size of wavelength, wavelength be 315～
The long wave ultraviolet (UVA) of 400nm, wavelength is the ultraviolet B radiation (UVB) of 280～315nm, and wavelength is 200～280nm
Short wave ultraviolet (UVC).But through ozone layer, most UVC, the UVB of up to 90% and the UVA of 10% are
Absorbed (Bode AM, and Dong Z.Mitogen-activated protein kinase activation in UV-induced
signal transduction.2003；Sci.STKE:RE2.doi:10.1126/stke.2003.167.re2).Time long
Between UVA expose, peroxide can be produced, cause the damage of DNA, UVB then can cause Skin Cell DNA photoadduct fourth
The formation of alkane pyrimidine dimer, affects the reparation after Skin Cell damage, causes the sudden change of DNA, cause the generation of skin carcinoma
(Kielbassa C, Roza L, and Epe B.Wavelength dependence of oxidative DNA damage
induced by UV and visible light.Carcinogenesis.1997；18:811-816.doi:10.1093/
carcin/18.4.811).Therefore, UVA and UVB is considered as the principal element causing dermal photodamage and skin carcinoma.Arrive
Reach the ultraviolet of earth surface to be mainly UVA (accounting for reaching the 95% of earth's surface SUV total amount) and a small amount of UVB and (account for reaching earth's surface SUV
The 5% of total amount).Although, people have done numerous studies for UVA and the UVB mechanism of action, but, people are to ultraviolet induction
Pathological change understand the most abundant.Ultraviolet radiation may result in oxidative stress, causes a series of scytitis, such as light
Linear skin is sick, psoriasis, even skin carcinoma (Svobodova A, Walterova D, and Vostalova J.
Ultraviolet light induced alteration to the skin.Biomed Pap Med Fac Univ Palacky
Olomouc Czech Repub.2006；150:25-38).Therefore, if the inflammatory signals causing photic damage can be suppressed to lead to
Road, it is possible to the dermal photodamage that effectively preventing ultraviolet radiation causes.P38, JNK path is cell transcription and translation
Major avenues of approach (Kaminska B.MAPK signaling pathways as molecular targets for
anti-inflammatory therapy--from molecular mechanisms to therapeutic benefits.
Biochim.Biophys.Acta.2005；1754:253-262.doi:10.1016/j.bbapap.2005.08.017；
Schindler JF, Monahan JB, Smith WG.p38 pathway kinases as anti-inflammatory drug
targets.J Dent Res.2007；86:800-811.doi:10.1177/154405910708600902；Huang P,
Han J, Hui L.MAPK signaling in inflammation-associated cancer development.Protein
Cell.2010；1:218-226.doi:10.1007/s13238-010-0019-9), ultraviolet irradiation can activate this path,
Cause generation (Hwang BM, Noh EM, Kim JS, Kim JM, You YO, Hwang JK, the Kwon KB, Lee of scytitis
YR.Curcumin inhibits UVB-induced matrix metalloproteinase-1/3 expression by
suppressing the MAPK-p38/JNK pathways in human dermal fibroblasts.Exp Dermatol.
2013；22:371-374.doi:10.1111/exd.12137).
Cefradine (Cephradine, Velosef), another name: cephazolin I, 11436 cefradine etc., narrow for the first generation
Become cephalosporin.This product is acidproof, and for wide spectrum, efficient, low toxicity antibiotic, feature is resistance to beta lactamase, to drug resistance gold Portugal
Bacterium and other multiple bacillus to broad ectrum antibiotic drug resistance etc. have bactericidal action rapidly and reliably, mainly with original shape through homaluria.
Clinic is mainly used in the infection of respiratory tract, urinary tract, skin and soft tissue etc..
TOPK, has another name called PBK, i.e. PDZ connects kinases and the cytotoxic T lymphocyte endogenous binding protein kinases of Lymphokine,
It is a kind of silk-threonine kinase (Abe Y, Matsumoto S, Kito K, et al.Cloning and expression of
A novel MAPKK-like protein kinase, lymphokine-activated killer T-cell-originated
Protein kinase, specificallyexpressed in the testis and activated lymphoid cells.
J Biol Chem 2000；275:21525-21531；Gaudet S, Branton D, Lue RA.Characterization
Of PDZ-binding kinase, a mitotickinase.Proc NatlAcad Sci 2000；97:5167-5172),
Belong to the newcomer of MAPKK (Mitogen-ativated protein kinase kinase) molecule families.It is p38 and
Upstream kinases (Abe Y, Matsumoto S, Kito K, the Ueda N.Cloning and expression of a novel of JNKs
MAPKK-like protein kinase, lymphokine-activated killer T-cell-originated protein
Kinase, specifically expressed in the testis and activated lymphoid cells.J Biol
Chem.2000；275:21525-21531.doi:10.1074/jbc.M909629199；Oh SM, Zhu F, Cho YY, Lee
KW, Kang BS, Kim HG, Zykova T, Bode AM, Dong Z.T-lymphokine-activated killer
cell-originated protein kinase functions as a positive regulator of c-Jun-NH2-kinase
1 signaling and H-Ras-induced cell transformation.Cancer Res.2007；67:5186-5194.
Doi:10.1158/0008-5472).TOPK can promote the mitosis of tumor cell, causes the damage of DNA, tumor cell
Transfer and generation (Gaudet S, Branton D, the and Lue RA.Characterization of of body inflammatory
PDZ-binding kinase, a mitotic kinase.Proc Natl Acad Sci USA.2000；97:5167-5172.
Doi:10.1073/pnas.090102397).There are some researches show, TOPK may participate in the DNA damage of ultraviolet induction, activates
P38, phosphorylation JNK (Liu K, Yu D, Cho YY, Bode AM, Ma W, Yao K, Li S, Li J, Bowden GT,
Dong Z, Dong Z.Sunlight UV-induced skin cancer relies upon activation of the p 38 alpha
signaling pathway.Cancer Res.2013；73:2181-2188.doi:10.1158/0008-5472).TOPK
Can with the H2AX in phosphorylation Ser139 site, the H2AX of phosphorylation then be detection DNA damage biological marker (Zykova TA,
Zhu F, Lu C, Higgins L, Tatsumi Y, Abe Y, Bode AM, Dong Z.Lymphokine-activated killer
T-cell-origihated protein kinase phosphorylation of histone H2AX prevents
arsenite-induced apoptosis in RPMI7951 melanoma cells.Clin Cancer Res.2006；12:
6884-6893.doi:10.1158/1078-0432；Kuo LJ, Yang LX.Gamma-H2AX-a novel biomarker for
DNA double-strand breaks.In Vivo.2008；22:305-309).Therefore, by finding a kind of drugs block
TOPK approach, can be that the preventing and treating preventing and treating UV-induced DNA damage and scytitis and tumor provides new possibility.
At present, people are only found that two kinds of TOPK inhibitor, HI032 and OTS514 (Kim DJ, Li Y, Reddy K, Lee
MH, Kim MO, Cho YY, Lee SY, Kim JE, Bode A, Dong Z.Novel TOPK inhibitor HI-TOPK-032
effectively suppresses colon cancer growth.Cancer Res.2012；15:3060-3068.doi:
10.1158/0008-5472；Matsuo Y, Park JH, Miyamoto T, Yamamoto S, Hisad S, Alachkar H,
Nakamura Y.TOPK inhibitor induces complete tumor regression in xenograft models of
human cancer through inhibition of cytokinesis.Sci.Transl.Med.2014；6:259ra145.
Doi:10.1126/scitranslmed.3010277).But these drug side effectes are obvious, it is impossible to by effective clinical practice.
In the present invention, we apply virtual screening based on structure to screen the medicine of FDA approval.Cefradine is criticized as by FDA
Quasi-first generation broad ectrum antibiotic, can be used to block TOPK path, the dermal photodamage reaction of suppression UV induction.
Summary of the invention
It is an object of the invention to:
1. find that cefradine can directly combine with TOPK.
2. find cefradine suppression TOPK activity, reduce p38, JNK and HA2X in TOPK and downstream signaling pathway
Phosphorylation level and inflammatory factor IL-6 and the secretion of TNF-α.
3. find that in the Photodermatoses that UV causes, TOPK and downstream kinase and inflammatory Cytokines Expression increase.
4. determine that cefradine prevention and/or treatment UV induction TOPK activity increase the Photodermatoses caused extremely.
The present invention is realized in
1. vitro kinase activity experiment, cefradine can directly combine with TOPK, suppression TOPK activation.
1) we apply homology modeling and molecular docking scientific discovery cefradine can combine with TOPK.Application molecular docking
Producing Binding Model after agonist, its display ATP with TOPK site combines, and cefradine utilizes two chains of E100 and G102
Formed after two hydrogen bonds therewith further combined with.The hydrophobic part of cefradine combines to the corresponding hydrophobic site of TOPK, and close
Outside water section is then exposed to.
2) application cefradine microgranule and HaCaT cell lysate carries out external binding tests and shows, cefradine can directly and
TOPK combines.
2. find cefradine suppression TOPK activity.
1) application cell survival rate test display cefradine will not reduce HaCat cell and the survival rate of JB6 cell.
2) cefradine can suppress TOPK and the activation of downstream passages kinases P38, JNK that UV induces, and depression effect is the time
-dose dependent.
3) utilizing GST-H2AX as substrate, be utilized respectively 0.5, the cefradine of 1,2mM combines with the TOPK of the state of activation
Carry out VITRO KINASE ASSAY.Result display HA2X phosphorylation level reduces with the increase of cefradine concentration during pretreatment.
4) cefradine causes, by combining UV in TOPK suppression HaCat cell and JB6 cell, the DNA that TOPK activation produces
Damage, show as cefradine by suppression TOPK to H2AX phosphorylation produce inhibitory action, and in time m-dose dependent.
5) ELISA result is shown, the cefradine of 2mM concentration significantly inhibits IL-6, TNF-α in HaCat cell and JB6 cell
Secretion, m-dose dependent when depression effect is.
In the Photodermatoses that 3.UV causes, TOPK expresses and increases.
1) the skin lesion sample HE dyeing display of (farmersskin) patient in the Photodermatoses that UV causes: epidermis angle
Changing excessively, epidermis thickens, edema, a large amount of cell infiltration.Showed by immune group result: P-P38, P-JNK, TOPK are at light
Expression in linear skin disease skin lesion is higher than normal skin.
2) UV irradiation HaCat cell and JB6 cell, in cell, P-P38, P-JNK all generate increase, in time m-dosage depend on
Lai Xing.
3) select shTOPK-2 and shTOPK-5 transfection TOPK cell strain to make TOPK gene silencing, find suppression TOPK gene
P38, JNK phosphorylation of UV induction in HaCaT cell can be suppressed.
4. cefradine prevention and/or treatment UV induction TOPK activity increase the Photodermatoses caused extremely.
1) the mouse skin inflammation that cefradine causes for UV irradiation produces inhibitory action.Is drawn materials in test position and carry out HE dye
Color, SABC and elisa assay show, cefradine external+SUV irradiation group is compared with SUV irradiation group, epidermal thickness,
Edema, cell infiltration degree substantially alleviate；P-P38, P-JNK, P-H2AX fall reaches 82%, 89% and 72% respectively；
Pro-inflammatory cytokine IL-6, the generation of TNF-α are substantially suppressed.
2) SABC pathology finds, after UV irradiation, in human skin tissue, p38, JNKs and H2AX phosphorylation level substantially rises
Height, after application cefradine, its phosphorylation level is substantially suppressed.
Result shows, cefradine external can be by suppressing internal TOPK and the activation of path thereof, and suppression pro-inflammatory cytokine is for skin
Skin damages and protects skin histology from the infringement of UV irradiation.
Present invention have the advantage that
1. present invention firstly discovers that cefradine can directly combine with TOPK.
Find cefradine suppression TOPK activity first, reduce p38, JNK and HA2X in TOPK and downstream signaling pathway
Phosphorylation level and inflammatory factor IL-6 and the secretion of TNF-α.
Find TOPK and downstream kinase and inflammatory factor in the Photodermatoses (farmersskin) that causes of UV first
Expression is increased.
Determine that cefradine prevention and/or treatment UV induction TOPK activity increase the Photodermatoses caused extremely first.
5. speculating according to above invention advantage, cefradine can prevent and/or treat UV abnormal the increasing of induction TOPK activity leads
Other inflammation (psoriasis, lupus erythematosus etc.) caused and tumor (cutaneous squamous cell carcinoma, basal cell carcinoma etc.).
Accompanying drawing explanation
Fig. 1: the technology of the present invention route map.
Fig. 2: cefradine combines TOPK and suppresses TOPK activity.
A. the docking model of the chemical constitution of cefradine and cefradine.Docking model describes with " material and method ".
B. cefradine is directly and the external combination of predecessor.Sepharose 4B is for combining and drop-down algoscopy.1st passage is input
Control (protein standard of TOPK).2nd passage is negative control, shows between TOPK and granule the most relevant.3rd passage
Road indicates TOPK and is combined with cefradine sepharose 4B granule.
C. cefradine suppresses TOPK activity in vitro in dose-dependent mode.Inactive GST-H2AX albumen is as having
The ATP substrate of activity TOPK and 100uM, this albumen 10%SDS-PAGE and the detection of Western blot method.
D. cefradine on the cell viability of HaCaT and JB6 cell do not affect.By HaCaT cell and JB6 cell head
Spore draws fixed 0.04, and 0.5,1 and 2mM processes 24 hours, and uses MTS to measure.Three independent measuring cells are lived
Power, result is meansigma methods ± SEM.
Fig. 3: UV induces downstream TOPK signal path with dosage and time dependence, and cefradine suppresses this signalling channel
Also suppress the secretion of cytokine in HaCaT cell and JB6 simultaneously.
A. by HaCaT cell (1 × 106) culture dish that is placed on 10 centimetres cultivates 24 hours, then by nothing in DMEM culture medium
Serum free culture system 12 hours.Then, carry out cell 1mM cefradine respectively 2,4,6,12 hours processing.Then place
40KJ/m is carried out at incubator2SUV process 5 minutes.Described cell lysate (30 microgram) carries out 10%SDS-PAGE and coagulates
Glue processes and the protein belt obtained is carried out Westernblot detection.
B. by HaCat cell (1 × 106The culture dish 24 hours at 10-cm is cultivated, then with depletion of blood in DMEM culture medium in)
Clear cultivating 12 hours, with serum-free culture 12 hours in DMEM culture medium, more respectively with 0.5, the cephalo of 1,2mM dosage draws
Determine to carry out processing 6 hours.Then cell carries out 5 minutes 40KJ/m at incubator2UV process.
C and D.JB6 cell processes equally with HaCat cell at identical conditions.HaCaT and JB6 cell is little through 24
Time Nature enemy after with 2mM6 hour cefradine cell pretreatment, then use 40KJ/m2SUV stimulates, and culture medium was at 24 hours
Collect.ELISA kit is used to measure IL6 and the concentration of TNF-α.The value of display is the data from three independent experiments
Mean value ± SEM.
The DNA damage of Fig. 4: cefradine suppression SUV induction, is induced by suppression SUV (having dosage and time dependence)
TOPK activation in HaCat cell and JB6 cell.
In A.HaCaT cell, TOPK is at 40KJ/m2SUV under conditions of H2AX phosphorylation level.HACAT siMock is thin
Born of the same parents and HACAT siTOK use 40KJ/m after within non-serum starved 12 hours, processing2SUV stimulate, irradiate latter 5 minutes extraction cell
Histone thing.The phosphorylation level of H2AX is detected by Western blot.
(B) there are dosage and time dependence during the phosphorylation level of H2AX during SUV stimulates HaCat cell and JB6 cell.Will
Cell Nature enemy 12 hours, by the process of SUV40KJ/m2 different time in incubator.Extract cellular histone and
Phosphorylation level by western blot method detection H2AX.
(C) cefradine suppression SUV induction H2AX is in the phosphorylation of HaCat and JB6 cell, and this process has time mediating recipe
The dependency of amount.By hungry for cell 12 hours, different time points with variable concentrations cefradine and to and 40KJ/m2SUV
Process.Cellular histone extracts and carries out 15%SDS-PAGE Gel Treatment, carries out the detection of Western blot method.JNKs
Can not combination external with cefradine.
Fig. 5: the phosphorylation overexpression of TOPK, p38 and JNK in people's Photodermatoses (farmersskin).
A. the pathological change of Photodermatoses is detected by H&E.
B. (B, C and D) phosphorylated p38 and phosphorylation JNK are at people's Photodermatoses (farmersskin) (N=8)
With in normal skin tissue (N=6), be the representational immunostaining results of TOPK.
Fig. 6: UV induction, in HaCaT cell and JB6 cell, has the phosphorus of p38 and JNK of dosage and time dependence
Acidifying.
A. HaCaT cell (1 × 106) is cultivated 24 hours in the culture dish of 10-cm, then use DMEM serum-free culture
Base is cultivated 12 hours, then irradiates with the SUV of various dose.Finally cell is changed incubator, after irradiating 5 minutes with SUV
Obtain result.
B. HaCaT cell (1 × 106) is cultivated 24 hours in the culture dish of 10-cm, then use DMEM serum-free culture
Base is cultivated 12 hours, then irradiates with the SUV of 40KJ/m2 dosage.Cell in incubator through the 40KJ/m2 of different time
The irradiation of SUV, then obtain another result.Above-mentioned cell lysate (30 μ g) carries out 10%SDS-PAGE.Obtain
Protein belt carry out Western blot method detection.
Fig. 7: suppression TOPK express with the phosphorylation of suppression UV induction p38 and JNK.
A. in HaCaT cell, effectively stop TOPK 2# and TOPK 5#.
B.TOPK 2# phosphorylation of suppression p38 and JNK in the case of UV irradiates is notable.HaCaT siMock cell and HaCaT
After hungry 12 hours of siTOK cell non-serum, stimulate with SUV light (40KJ/m2).Extract after SUV light irradiates 5 minutes
Take full cell lysate.The phosphorylation level of P38 and JNK is carried out Western blot detection.(C) TOPK 5# is stoped
Notable to the phosphorylation of suppression SUV induction p38 and JNK.
The inflammation of Fig. 8: cefradine suppression SUV radiation-induced mouse skin.
A. the epidermis of cefradine suppression SUV induction thickens the infiltration with inflammatory cell.BABL/c mice cefradine of growing up is coated with the back of the body
Portion's skin 3 hours, through 100KJ/m2SUV irradiate, draw materials after irradiating latter 24 hours, carry out HE and IHC.
B. p38, JNKs and the H2AX in mice skin tissue phosphorylation quantified, by Image-ProPlus software detection,
Data are shown as Integrated optical densities unit.The value of display is mean value ± SEM of the data from three independent experiments.With the most right
Compare significantly according to group, P ＜ 0.01.Notable P ＜ 0.05 is contrasted with simple SUV group.
C.SUV100KJ/m2Under irradiation, IL-6 and the secretion of TNF-α in cefradine suppression mice skin tissue.Use ELISA
Kit measurement IL-6 and the concentration of TNF-α.Significant difference is determined by the one-way analysis of variance.Compared with simple matched group
Significantly, P ＜ 0.01.Notable with simple SUV contrast, P ＜ 0.05.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described in detail.It should be noted that embodiments of the invention are only limitted to this
Invention illustrates, and does not limit effect.Relevant test method involved in embodiment and other various experimental implementation, all
For the ordinary skill in the art, the part being not particularly illustrated in literary composition, those of ordinary skill in the art is referred to Shen of the present invention
Please various common tool books, scientific and technical literature or relevant description, handbook etc. before day be practiced.
Embodiment one cefradine combines TOPK and suppresses TOPK activity.
(A) cefradine reagent is bought in Sigma-Aldrich company (St Louis, MO, USA).The mankind activate TOPK
Kinases is purchased from Millippore (Billerica, MA, USA).pGEX-GST-H2AX、lentiviral expression、
Packaging vectors:TOPK shRNA, pMD2.0G and psPAX are purchased from Addgene company.IL6、TNF-α ELISA
Determination box be purchased from Biosen company (Beijing. China).Internal reference albumen purchases Santa Cruz Biotechnology (Santa
Cruz, CA, USA).
Molecular model and docking simulation
In order to estimate the interaction model of TOPK and cefradine, model a TOPK structure, and be applied to follow-up induction
Agree with docking.The sequence of TOPK (GI:83305809), from the download of NCBI, carries out BLAST for albumen database (PDB)
Similarity retrieval is to differentiate homologous sequence.Have in the protein of highest serial homogeneity (30%) at those with TOPK, right
It is comprehensive that the structure of 4L52,2EVA and 4GS6 (PDB entry) has carried out protein-ligand, and it is multiple that this is applicable to TOPK-cefradine
The modeling of compound.The supernumerary structure of PDB 2F4J and TOPK have the sequence iden of 27%, template group based on previous studies
Chosen.The sequence of TOPK and four templates, 4L52,2EVA, 4GS6 and 2F4J, coupling ClustalW 2 server just
Use default parameters.The secondary structure of TOPE and template is predicted, and compares with SSpro4.0 server.Then,
Multiple Sequence Alignment is for setting up by MODELER 9v8 homology model.Generate ten models, optimal discrete optimization protein energy
(DOPE) structure of score is selected as TOPK modeling structure.This model TOPK reliability of structure, uses server S AVES
Confirm.Then copy TOPK structure great master 9.0 editions (Maestro version 9.0, Schrodinger Co., Ltd, 2010
Year, New York, New York) optimize in have submitted " protein formulation guide " module.The structure of cefradine is delineated and preparation makes
By the LigPre module of structure great master, its pH value is in the range of 7.0 ± 0.2.For the binding pattern generated, structure is big
The method of the induced-fit docking that teacher is 9.0 editions is used for residue and selects to remain flexibly the ATP binding pocket of TOPK.
External binding assay
HaCat cell pyrolysis liquid (1mg) is cultivated at cefradine-sepharose 4B or sepharose 4B in reaction buffer [50 mmoles
You are Tris (pH value 7.5), the ethylenediaminetetraacetic acid (EDTA) of 5mM, 150 mMs of NaCl, 1 mM of DTT, and 0.01%
NonidetP-40,2 mcg/ml bovine serum albumin, 0.02 milli Phenylmethanesulfonyl fluoride (PMSF) and 1 microgram/ml
Protease inhibitor mixes] in.At a temperature of 4 DEG C, jog overnight, will sway washing five times, SDS-PAGE electrophoresis, carry out afterwards
Western blot analyzes.
(C) the docking model of the chemical constitution of cefradine and cefradine.Docking model is described with " material and method ".Head
Spore draws the surely direct and external combination of predecessor.Sepharose 4B is for combining and drop-down algoscopy.1st passage is input control (TOPK
Protein standard).2nd passage is negative control, shows between TOPK and granule the most relevant.3rd passage road indicates TOPK
Be combined with cefradine sepharose 4B granule.
(D) cefradine suppresses TOPK activity in vitro in dose-dependent mode.Inactive GST-H2AX albumen is as having
The ATP substrate of activity TOPK and 100uM, this albumen 10%SDS-PAGE and the detection of Western blot method.
(E) cefradine on the cell viability of HaCaT and JB6 cell do not affect.By HaCaT cell and JB6 cell head
Spore draws fixed 0.04, and 0.5,1 and 2mM processes 24 hours, and uses MTS to measure.Three independent measuring cells are lived
Power, result is meansigma methods ± SEM.
Embodiment two SUV induces downstream TOPK signal path with dosage and time dependence, and cefradine suppresses this signal
Also suppress the secretion of cytokine while passage in HaCaT cell and JB6.
(A) cell is cultivated and cell proliferation rate detects
Human epidermal keratinocyte immortalized cell line strain, mouse skin JB6 Cl41 cell strain (JB6) is bought in ATCC,
Virginia, USA.HaCaT using DMEM as culture medium (including 100u/ml penicillin and 100u/ml streptomycin, 10%FBS),
It is placed on 37 DEG C, the incubator of 5%CO2 saturated humidity.JB6 cell strain, will using MEM as culture medium (including 5%FBS)
It is placed in 37 DEG C, the incubator of 5%CO2 saturated humidity.After blood counting chamber counting, by two kinds of cells with 1 × 105/ml's
Concentration is inoculated in the culture dish of 6cm.
Cell is inoculated in 96 well culture plates with the concentration of 8 × 103/ml, incubated overnight.Then, change culture fluid, will
The cefradine of various dose is configured to variable concentrations, adds fresh medium, cultivates cell.In different time points, apply MTS
Method measures cell proliferation vigor.Microplate reader is used to measure the absorbance in each hole at 490nm.
Western blot test
Cell is inoculated in 10cm culture plate with 1 × 106/ml, cultivates cell growth and be fused to 60%-70%.Cell is in nothing
Hungry cultivation 12 hours in blood serum medium.Then UV predose adds the cefradine of variable concentrations (0.1,0.2,0.5mM)
Cultivate 2,4,6,12 hours.Cell lysis, extracts albumen with Bradford method.20-30 μ g loading albumen adds 5 × SDS
Sample-loading buffer, will be by sample in 95 DEG C of albuminous degenerations 10 minutes before loading.Carry out SDS-PAGE electrophoresis, transferring film, PVDF afterwards
Film 4 DEG C of overnight incubation of monoclonal antibody, carry out antibody hybridization and luminescence, development treatment afterwards.
(B) culture dish that HaCaT cell (1 × 106) is placed on 10 centimetres is cultivated 24 hours, then with in DMEM culture medium
Serum-free culture 12 hours.Then, carry out cell 1mM cefradine respectively 2,4,6,12 hours processing.Then put
Put incubator carry out 40KJ/m2 SUV process 5 minutes.Described cell lysate (30 microgram) carries out 10%SDS-PAGE
The protein belt obtained also is carried out Westernblot detection by Gel Treatment.
(C) by HaCat cell (1 × 106) is cultivated the culture dish 24 hours at 10-cm, then by nothing in DMEM culture medium
Serum free culture system 12 hours, with serum-free culture 12 hours in DMEM culture medium, more respectively with 0.5, the cephalo of 1,2mM dosage
Draw and determine to carry out processing 6 hours.Then cell carries out the 40KJ/m of 5 minutes at incubator2The process of SUV.
(D) JB6 cell processes equally with HaCat cell at identical conditions.HaCaT and JB6 cell was through the famine of 24 hours
Starving and use 2mM6 hour cefradine cell pretreatment after processing, then stimulate with 40KJ/m2SUV, culture medium was collected at 24 hours.
ELISA kit is used to measure IL6 and the concentration of TNF-α.The value of display is the mean value ± of the data from three independent experiments
SEM。
The DNA damage of embodiment three cefradine suppression SUV induction, by suppression SUV (having dosage and time dependence)
The TOPK of induction activation in HaCat cell and JB6 cell.
(A) expression of antibacterial GST-H2AX fusion protein and purification
Mankind's GST-H2AX fusion protein is expressed in E.ColiBL21 antibacterial.When 37 DEG C, antibacterial under the wavelength of 600nm,
Growing with the absorbance of 0.8～0.9, after 2-3 hour, induction produces IPTG, afterwards centrifugal extraction.Cell granulations is suspended in
In PBS liquid.After centrifugal degradation, supernatant and 4 DEG C of night incubation of Glutathione Sepharose, used PBS afterwards,
Then contact with 50mM glutathion.After protein quantification, with SDS-PAGE electrophoresis, develop the color with Coomassie brilliant blue.
External measuring method for activity
With GST-H2AX (1 μ g) albumen as substrate, carry out experiment in vitro with the TOPK of the 250ng state of activation.Both are containing
In the kinase buffer liquid of 100 μMs of ATP, 30 DEG C are reacted 30 minutes, terminate reaction with the sample solution of 5 × SDS afterwards.P-H2AX、H2AX
All it is measured with Western with all of TOPK.
(B) in HaCaT cell, TOPK H2AX phosphorylation level under conditions of the SUV of 40KJ/m2.HACAT siMock
Cell and HACAT siTOK use 40KJ/m after within non-serum starved 12 hours, processing2SUV stimulate, irradiate extraction in latter 5 minutes thin
Born of the same parents' histone thing.The phosphorylation level of H2AX is detected by Western blot.
(C) there are dosage and time dependence during the phosphorylation level of H2AX during SUV stimulates HaCat cell and JB6 cell.Will
Cell Nature enemy 12 hours, by SUV 40KJ/m in incubator2The process of different time.Extract cellular histone and
Phosphorylation level by western blot method detection H2AX.
(D) cefradine suppression SUV induction H2AX is in the phosphorylation of HaCat and JB6 cell, and this process has time mediating recipe
The dependency of amount.By hungry for cell 12 hours, different time points with variable concentrations cefradine and to and 40KJ/m2SUV
Process.Cellular histone extracts and carries out 15%SDS-PAGE Gel Treatment, carries out Westernblot method detection.JNKs
Can not combination external with cefradine.
TOPK and p38, the phosphorylation overexpression of JNK in embodiment four mankind's Photodermatoses skin lesion.
(A) dyeed by mankind Photodermatoses skin lesion tissue pathological slice HE, observe the pathological change of Photodermatoses.
(B) mankind's Photodermatoses skin lesion histogenic immunity histochemical staining observing.TOPK used in experiment, JNKs, p38,
H2AX, P-JNKs (Thr183/Thr185), P-P38 (Thr180/Thr182), P-H2AX (Ser139) antibody all purchase in
Cell Signaling Technology (Billerica, MA, USA).Tissue slice (5 microns) first dewaxes and citric acid
Microwave is used to carry out antigen retrieval after the fluid infusion in 10 minutes of sodium buffer.Section is cooled to room temperature, by the hydrogen peroxide treatment of 3%
10 minutes, and use 5% lowlenthal serum, at room temperature 40 minutes.Next step, be incubated overnight section primary antibody at 4 DEG C.
Then section is washed in PBS, and with the second antibody incubation together of 30 minutes.After PBS washing, will section and 3,3-
Diaminobenzidine (DAB) is as 3 minutes incubations of substrate.For evaluation result, use digital camera photomicrograph.To each
Positive stained cells in microphotograph counts.Phosphorylated p38 and phosphorylation JNK are at mankind's Photodermatoses (N=8)
Compare with normal skin tissue (N=6) and occur increasing, be the representative immunostaining results that increases of TOPK activity.
Embodiment five suppresses TOPK's to express the phosphorylation inducing p38 and JNK with suppression UV.
(A) in HaCaT cell, TOPK2# and TOPK5# is effectively stoped.
(B) TOPK2# phosphorylation of suppression p38 and JNK in the case of UV irradiates is notable.HaCaT siMock cell and HaCaT
After hungry 12 hours of siTOK cell non-serum, with SUV light (40KJ/m2) stimulate.After SUV light irradiates 5 minutes, extraction is complete
Cell lysate.The phosphorylation level of P38 and JNK is carried out Western blot detection.
(C) stop TOPK5# notable to the phosphorylation of suppression SUV induction p38 and JNK.
Embodiment six SUV induces in HaCaT cell and JB6 cell, has p38 and JNK of dosage and time dependence
Phosphorylation.
(A) by HaCaT cell (1 × 106) cultivate 24 hours in the culture dish of 10-cm, then use DMEM serum-free culture
Base is cultivated 12 hours, then irradiates with the SUV of various dose.Finally cell is changed incubator, after irradiating 5 minutes with SUV
To result.In experiment, Q-Lab company (Cleveland, OH) purchased by SUV lamp (UVA-340nm).In SUV, UVA, UVB spoke
According to respectively accounting for 92.5%, 7.5%, measure its dosage with supporting measuring instrument.
(B) by HaCaT cell (1 × 106) cultivate 24 hours in the culture dish of 10-cm, then use DMEM serum-free culture
Base is cultivated 12 hours, then with 40KJ/m2The SUV of dosage irradiates.Cell in incubator through the 40KJ/m of different time2's
The irradiation of SUV, then obtains another result.Above-mentioned cell lysate (30 μ g) carries out 10%SDS-PAGE.The egg obtained
Peduncle carries out the detection of Western blot method.
The inflammation of embodiment seven cefradine suppression SUV radiation-induced mouse skin.
(A) animal experiment study
6-8 week old grow up Balb/c mice be from Center for Disease Control's (Hubei China) buy.Make the normal natural conditions of animal
Under conditions of, have a rest 5 days.The back hair testing first 24 hours animals is shaved, and is coated with cefradine without hair skin of back,
Resting stage 100KJ/m2SUV irradiate 3 hours.Euthanasia mice also collects its back body skin sample.The half of sample
It is fixed on 4% paraformaldehyde and HE dyeing (H&E) dyeing and immunohistochemical staining (IHC) immediately.Other sample is cold
Freeze and for elisa assay.All laboratory animals are in compliance with the relevant regulations of National Laboratory Animal.
(B) epidermis of cefradine suppression SUV induction thickens the infiltration with inflammatory cell.Adult BABL/c mice cefradine
It is coated with skin of back 3 hours, through 100KJ/m2SUV irradiate, draw materials after irradiating latter 24 hours, carry out H&E and IHC.
(C) p38, JNKs and the H2AX in mice skin tissue phosphorylation quantified, is examined by Image-ProPlus software
Surveying, data are shown as Integrated optical densities unit.The value of display is mean value ± SEM of the data from three independent experiments.With list
Pure matched group is compared significantly, P ＜ 0.01.Notable P ＜ 0.05 is contrasted with simple SUV group.
(D)SUV 100KJ/m2Under irradiation, IL-6 and the secretion of TNF-α in cefradine suppression mice skin tissue.Use ELISA
Kit measurement IL-6 and the concentration of TNF-α.Significant difference is determined by the one-way analysis of variance.Compared with simple matched group
Significantly, P ＜ 0.01.Notable with simple SUV contrast, P ＜ 0.05.
By above example, in conjunction with the Molecular biological function in terms of the physiology of TOPK, pathophysiology, it can be seen that remove
Cefradine is by combining and suppressing TOPK activity to prevent and/or the abnormal skin increased for the treatment of TOPK activity in the present invention
Inflammation, outside the Photodermatoses (sunburn and farmersskin) of UV induction, thus it is speculated that cefradine can also lead to
Cross and combine and suppress the activity prevention of TOPK and/or treat abnormal other scytitiss increased of TOPK activity and tumor, as respectively
Psoriasis pustulosa, lupus erythematosus, daylight seborrheic keratosis, melanoma, Skin Squamous Cell Carcinoma etc., and human body other system organ-tissue TOPK
The abnormal inflammation increased of activity and tumor.Simultaneously, thus it is speculated that the dosage that cefradine is bigger, it is possible to create preferably effect.
Claims (9)
1. the pharmacological action that cefradine (Cephradine, Velosef) is new, it is characterised in that cefradine can
Directly be combined with TOPK and reduce its activity.
2. the pharmacological action that cefradine described in claim 1 is new, it is characterised in that cefradine prevents and/or controls
Treat the abnormal inflammation increased of TOPK activity.
3. the pharmacological action that cefradine described in claim 1 is new, it is characterised in that
1) cefradine can directly be combined with TOPK.
2) producing Binding Model after application molecular docking agonist, display ATP with TOPK site combines, cephalo
Draw surely utilize after E100 and G102 two hydrogen bonds of two chain formation therewith further combined with.Dredging of cefradine
Water section combines, outside hydrophilic segment is then exposed to the corresponding hydrophobic site of TOPK.
3) cefradine microgranule and HaCaT cell lysate carry out external combination.
4. the pharmacological action that cefradine described in claim 1 is new, it is characterised in that cefradine reduce TOPK and
The phosphorylation level of p38, JNK and HA2X and inflammatory factor IL-6 and TNF-in downstream signaling pathway
The secretion of α.
5. the inflammation described in claim 2 includes Photodermatoses, it is characterised in that UV cause actinic
In dermatosis, TOPK expresses and increases.
6. the TOPK described in claim 5 expresses and increases, it is characterised in that people's Photodermatoses that UV causes
In skin lesion, in TOPK and downstream signaling pathway, P38, JNK of phosphorylation express and increase.
7. cefradine described in claim 2 is new prevention and/or the application for the treatment of inflammation, it is characterised in that:
1) cefradine prevention and/or treatment UV induction TOPK activity increase the Photodermatoses caused extremely.
2) carry out UV irradiation after application cefradine, mouse skin edema, thicken, the inflammation such as cell infiltration degree
Disease pathological manifestations substantially alleviates；P38, JNK in TOPK that in skin lesion, UV irradiation causes and downstream signaling pathway
Phosphorylation raises and inflammatory factor IL-6, and the secretion of TNF-α increases and substantially suppressed.
3) after being application cefradine, TOPK and downstream signaling pathway in the human skin tissue that UV irradiation causes
The rising of middle p38, JNK and H2AX phosphorylation level is substantially suppressed.
8. the Photodermatoses described in claim 5-7, is characterized in that in a few days tanning severely and farmersskin.
9. the effective cell concentration of the cefradine suppression TOPK activity described in claim 7 is at 0.1mM to 2mM
Between.
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| CN108066345A (en) * | 2016-11-14 | 2018-05-25 | 武汉华杰世纪生物医药有限公司 | A kind of compound with antitumor action |
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| WO2007099396A2 (en) * | 2005-06-07 | 2007-09-07 | Foamix Ltd. | Antibiotic kit and composition and uses thereof |
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